1973
V O L U M E 28, NO. 12, D E C E M B E R 1 9 5 6 Table 11. Determination of Hydrogen Chloride in Presence of .4cid Chlorides Hydrogen Chloride ,% -4cid Chloride Added Found 2.02 2.07 Acetyl chloride 3.85 6.14 16 50 1.95 3.49 6.72 14.72 0.72 1.67 3.08 4.00 1.24 2 35 3.45 7 88 1 42 2 64 5 10 10 53 1 14 2 10
Propionyl chloride
Lauroyl chloride
Paliiiitoyl chloride
Benzoyl chloride
Saphthoyl chloride Fatty acid chloride (av. mol. wt. = 293.5)
0 11
0 81 1 87
3.93 6.30 17.08 1.94 3.65 6.19 14.47 0.70 1.65 3.24 4.21 1.37 2 47 3.60 8 49 1 57 2 75 5 17 10.64 1 21 2 14 0 13 0 80 1 83
Table 111.
Analysis of Succinyl and Naphthoyl Chlorides Hydrogen Acid Carboxylic Chloride, Chloride, Acid, dcid Chloride 70 % % Succinyl chloride 0.0 99.4 ..
Naphthoyl chloride
0.0
99.4 99.1 99 1 99.2 99 2 98.9 93.6 93.7 93.6
..
..
used for 2 months or more without redistillation. The acetone employed contained about 0.2% water, but the acid chloridcs react rapidly enough x i t h the m-chloroaniline to prevent any hydrolysis from taking place. Acetone dried over Drierite gave the same results as undried acetone. Synthetic mixtures of acid chlorides and the corresponding acids were prepared and analyzed by Procedure B (Table I ) and synthetic mixtures of acid chlorides and hydrogen chloride \? eie analyzed by Procedure A (Table 11) t o test the method. The acid chlorides used in these mixtures were distilled from the best available samples to obtain acid chlorides free of hydrogen chloride and carboxylic acid. Because of the difficulty of adding known amounts of dry hydrogen chloride directly to acid chlorides to obtain synthetics of known composition, the hydrogen chloride-acid chloride synthetics were prepared by adding aliquots of an ether solution of hydrogen chloride of known composition to weighed samples of acid chlorides. Results obtained for succinyl chloride and naphthoyl chloi ide are given in Table 111. S o synthetic mixtures were prepared for these compounds, because in the case of succinyl chloride the second chloride group reacts incompletely and the free carboxylic acid values obtained cannot be used. The unreacted acid chlcride group forms carboxylic acid 11 hen n ater is added, and high results are obtained for free carboxylic acid. I n the caw of naphthoyl chloride the authors were unable to obtain a priie sample from which to prepare synthetic mixtures. The analysis of three acid bromide samples was attempted, but good results were obtained only for benzoyl bromide. In the analysis of benzoyl bromide 25 ml. of ether were used in Procedure B instead of 25 ml. of acetone. Low results for acid bromide were obtained when acetone was used, Results obtaincd for acetyl and valeryl bromides varied greatly.
..
..
..
.. 6 3 6.3 6 3
DISCUSSION AND RESULTS
All reagents were used %E obtained, except the m-chloroaniline, r h i c h was distilled. After distillation the m-chloroaniline can be
LITERATURE CITED
Ackley, R. R., Tesoro, G. C., ISD. ENG.CHEM.,ANAL. ED. 18, 444-5 (1946).
Drahowzal, F., Klamann, D., Monatsh. 82, 470-2 (1951). Fritz, J. S.,ANAL.CHEM.23, 589-91 (1951). Klamann, D., Monatsh. 83, 719-23 (1952). Mitchell, J . , Smith, D. XI.,“Chemical Analysis,” Vol. V, “Aquaiiietry,” pp. 369-71, Interscience, Xew York, 1948. Pesee, AI., Willemart, R., Bull. SOC. chim. France 1948, 479-80. Usanovich, hl., Yatsimirshii, K., J . G‘en. Chem. (U.S.S.R.) 11, 957-8 (1941). RECEIVED for review May 24, 1956.
Accepted August 10, 1956.
Colorimetric Determiiatien of Biphenyl in Biological ROBERT
B. BRUCE
and JOHN W. HOWARD
Hazleton Laboratories, Falls Church, Va.
A colorimetric method has been devised for the determination of biphenyl in biological materials. Biphenyl is nitrated in acetic acid, using concentrated nitric acid. The product is shown to be 4-nitrobiphenyl, The nitro derivative is subsequently reduced and the resulting amine coupled with N-(1-naphthy1)ethylenediamine to give a purple dye. The absorbance of this solution is proportional to the concentration in the range of 10 to 100 y. This method has been successfully used in the study of the metabolism of biphenyl.
D
URIXG the investigation of the metabolism of biphenyl it was necessary to have a method suitable for its determination in biological materials. Among the methods available in the literature(I-Q), that of Dickey and Green ( 1 )appeared
to offer the best advantages for the determination of biphcm\I in biological materials. However, on treating urine by this method extremely high blanks were obtained which could not easily be eliminated. A method which has proved to be satisfactory involves separation of the biphenyl with subsequent nitration to 4-nitrobiphenyl, reduction to the corresponding amine, and colorimetric determination of the amine by diazotization and coupling with AT-(1-naphthy1)ethylenediamine. This procedure has been used in this laboratory and elsewhere (6) for several months with satisfactory results, ANALYTICAL PROCEDURE
Reagents. Reagent grade chloroform, glacial acetic acid, rcagent grade nitric acid, and reagent grade sodium sulfate, conforming to ACS specifications.
1974
ANALYTICAL CHEMISTRY
Sodium carbonate, 5% (w,/v.) in distilled water Ethyl alcohol, 95% Sodium hydroxide, 5iz' Hydrochloric acid, reagent grade Potassium permanganate, 6% (w,/v.) in distilled water Zinc dust, certified reagent grade Sodium nitrite, 0.25 gram of reagent grade sodium nitrite in water to make 100 ml. Make up fresh daily. Ammonium sulfamate, 2.5 grams of technical ammonium sulfamate in water to make 100 ml. Make up fresh weekly. LV-(1-Saphthy1)ethylenediamine dihydrochloride, Eastman Xodak Co., 1 gram in water to make 50 ml. Make up fresh weekly. Biphenyl. Standard Solution A, 0.1000 gram of biphenyl in chloroform to make 100 ml. 1 ml. = 1000 micrograms of biphenyl. Standard Solution B, 2 ml. of Standard Solution A diluted to 100 ml. with chloroform. 1 ml. = 20 y of biphenyl. Apparatus. A Beckman Model DU spectrophotometer was used with 1.000-em. cuvettes. Test tubes, 22 by 200 mm., fitted with bulb condenser Volumetric flasks, 50-ml. and 100-ml. Soxhlet apparatus Centrifuge tubes, 50-ml., fitted rrith glass stoppers PREPARATION OF STANDARD CURVE
Aliquots of 0.5, 1.0,2.0, 3.0, and 4.0 ml. of Standard Solution B are run into 22 X 200 mm. test tubes, 0.5 ml. of glacial acetic acid is added, and the chloroform is evaporated a t 50' C. under a gentle stream of air to 1 or 2 ml. or less. Then 0.5 ml. of concentrated nitric acid is added, the solutions are mixed well, and the tubes are placed in a water bath at 70" C. for 1 hour. The nitration is st,opped by addition of 5 ml. of ice water. Two milliliters of chloroform are added to each tube and the nitro derivative is extracted into the chloroform by shaking the tubes for 2 to 3 minutes. The acid layer is drawn o f f and dkcarded. The tubes are placed in a warm water bath (50" to 60" C.) and the chloroform layer is evaporated under a gentle stream of air. One milliliter of 95% ethyl alcohol and 2 ml. of 5N hydrochloric acid are added and the tubes are shaken well to wash down the sides. Then 0.2 gram of zinc dust is added and the tubes are placed in a boiling water bath for 15 minutes, Each tube is fitted with a small bulb condenser. After reduction, the contents of each tube are filtered through Whatman S o . 42 paper into a 50-ml. volumetric flask. The condenser arid tube are well rinsed with water and the rinses are added to the flask to make the volume 40 ml. One millilit,er of 0.25% sodium nitrite is added to each Aask and the solution is mixed well and allowed to stand 10 minutes. One milliliter of 2.5% ammonium sulfamate is added to each solution, which is again mixed well and allowed to stand 10 minutes more. Finally, 2.0 ml. of 1% N-(1-naphthy1)ethylenediamine dihydrochloride are added to each solution and the solutions are made to 50 ml., mixed, and allowed to stand 1 hour for full development of the purple color. The absorbance is read at 570 inp, using :t blank solution prepared by carrying 2.0 ml. of tahloroform through the entire procedure.
Urine. Five milliliters of urine are pipetted into a glass-stoppered 50-ml. centrifuge tube and shaken for 2 to 3 minutes with 10 ml. of chloroform. After removal of the urine layer, the chloroform layer is washed with water, followed by a wash with 10 ml. of 5 N sodium hydroxide. The remainder of the procedure is thc same as for the blood analysis. Centrifugation may be necessar). in many of these steps, for adequate separation of layers. Feces. Five grams of a well-mixed sample of feces are placed in a mortar, followed by a portion of anhydrous sodium sulfate; the feces and sodium sulfate are adequately mixed until the fecal sample is in a dry state. The sample is then quantitatively transferred to a Soxhlet cup and extracted with chloroform for 3 hours. The extract is transferred to a 100-ml. volumetric flask and adjusted to volume with chloroform. .4 suitable aliquot for analysis is pipetted into a glass-stoppered centrifuge tube. The aliquot is shaken with 5 N sodium hydroxide for 2 to 3 minutes and the procedure as described for the blood and urine is continued. DISCUSSION
The absorbance curve of the colored compound formed by carrying 69 y of biphenyl through the procedure is presented in Figure 1. Maximum absorption lies a t 570 mp. An absorbanceconcentration curve determined for 10 t o 100 y of biphenyl showed that the Beer-Lambert relationship is followed over this range. Recoveries of known amounts of biphenyl added to samples of blood, urine, and feces (Table I) appear to be satisfactory. The method described is empirical in nature and each step should be followed exactly as given. However, some investigation was made of the reactions that occur during the nitration and subscquent reduction of thp biphenyl.
*
0.100
p.od
400
45 0
500
550
SO0
MILLlMlCRONS
DETERMINATION O F BIPIIENY L IN BIOLOGICAL !MATERIAL
Figure 1. Absorption curve
Blood. Five milliliters of blood (oxalated) are pipetted into a 22 x 200 nim. test tube, shaken for 2 to 3 minutes with IO ml. of chloroform, and then filtered through glass wool (wetted with vhloroform) into a 50-ml. glass-stoppered centrifuge tube. The test tube and precipitate are washed well with additional small ali uots of chloroform. ?he chloroform layer is washed with 10 ml. of 5 N sodium llydroxide by shaking for 2 to 3 minutes. The water layer is drawn off and the chloroform layer is washed twice more viith a 10-ml. portion of water. The chloroform layer is then washed with 10 ml. of 6yopotassium permanganate plus 5 ml. of glacial acetic- acid. For this 11-ashthe tubes are mechanically shaken for 1 hour. After this the permanganate layer is drawn o f f and the chloroform 1:tyer is shaken with 20 ml. of 5% sodium carbonate for 2 minutes. The carbonate layer is removed and the chloroform layer is washed twice again x i t h 20 ml. of water. After removal of the water layer, the chloroform layer is filtered wool (wetted with chloroform) into a 22 X 220 mm. h adequate rinsing, 0.5 ml. of glacial acetic acid is added, and the chloroform is evaporated to 1 or 2 ml. in a water bath a t 50" to 55' C. under a stream of air. The remainder of the procedure is the same :ts for preparation of the standard curve.
One gram of biphenyl was dissolved in 5 ml. of chloroform, and 5 ml. each of glacial acetic and nit'ric acids were added. The procedures described above were followed with proportionate increases in the amounts of reagents. After ext,raction of the nitro derivative into chloroform and washing, the chloroform extract was evaporated to dryness and the residue recrystallized from alcohol. The melting point of this compound was 113' C., corresponding to that of 4-nitrobiphenyl. The yield was well above 90% based on this derivative. Reduction of this nitro derivative by the methods described in the procedure for analysis and subsequent acylation with acetic anhydride gave a product with a melting point of 171' C., corresponding to that of .Vacetylbiphenylamine. This evidence indicates t,hat the primary product of nitration under the conditions of the analytical procedure is the mononit,ro derivative. Samples containing known amounts of 4nitrobiphenyl- and 4-hiphen!.lamine were carrid through the analytical procedure,.
V O L U M E 2 8 , NO. 12, D E C E M B E R 1 9 5 6 Table I.
Recoveries of Biphenyl Added to Blood, Urine, and Feces (5.0 ml. of blood and urine, and 5 grams of feces) Biphenyl, y
hdded
Recovered In Blood
10 20 25 40
9.2 18.5 22.7 40.5
20 25 40 40 50 75
I n Urine 17.2 30.4 41.4 40.3 44.0 77.4
Added 2 4 5 7
50
Biphenyl, M g . Recovered 2.30
% recovery
Av.
92 93 91 101 94
47,.
86 121 103 101 88 103 100
yo recovery 90
00 00 50
5 00
10.0
1975
I n preliminary work i t was found that during the evaporation of chloroform solutions of biphenyl considerable and variable amounts of biphenyl mere lost. The addition of acetic acid prior to evaporation prevented this loss. To solutions containing 5 and 10 7 of biphenyl in 10 ml. of chloroform, 0.50 ml. of acetic acid was added. The solutions were evaporated under gentle air streams again until no odor of chloroform remained. The resulting acetic acid solutions were diluted to 10.0 ml. with iso-octane and their absorbances compared a t 248 mp with that of pure biphenyl in iso-octane containing the same concentrations of acetic acid. The absorbances indicated that no biphenyl had been lost in the evaporation steps despite the marked volatility of biphenyl. The procedure used for purification of extracts of biological tissues is similar to that described by Dickey and Green (1). The preliminary wash with sodium hydroxide is intended to remove phenols that may have formed from metabolism of biphenyl. I n order to determine the efficiency of this wash, solutions containing 1 mg. each of 0- and p-phenylphenol and 4,4'dihydroxybiphenyl were carried through the procedure. No color was formed with any of these phenols. Control samples of blood, urine, and feces carried through the procedure gave only small blank readings. Although these were negligible, corrections were made. LITERATURE CITED (1) Dickey, E. E., Green, J.
beginning a t the appropriate steps. The absorbances per micromole were 0.875 for the nitro derivative and 0.872 for the amino derivative. The corresponding absorbance found when biphenyl was carried through the procedure was 0.705. These results indicate that approximately 20% is lost either during or hcfore nitration.
W., Fourdrinier Kraft Board Institute,
Appleton, Wis., Project 1108-7-4 (1955). ( 2 ) Kirchner, J. G., Mller, J. 31., Rice, R. G., J . Agr. Food Chem. 2, 1031 (1954). (3) Newhall, W.F., Elvin, E. J., Knodel, L. R . , ;IXAL. CHExr. 26, 1234 (1954). (4) Tomkins, It. G., Isherwood, F. A . , Analyst 70, 330 (1945). (5) W e s t , H. D., Meharry Medical College, Nashville. Tenn., personal communication. RECEIVEDfor review April 2 5 , 1956.
Accepted August 9 , 1956.
Use of 2,4=Dinitrophenylhydrazones of p-Phenylphenacyl Esters as Second Derivatives in Identification of Organic Acids HAWKINS NG, A. DINSMOOR WEBB, and RICHARD E. KEPNER Departments of Chemistry and of Viticulture and Enology, University of California, Davis, Calif.
The 2,4-dinitrophenylhydrazones of the p-phenylphenacyl esters of 18 fatty acids were prepared. In several cases a greater difference was observed between the melting points of the hydrazones than between the melting points of the correspondingesters. The double derivatives of the straight-chain saturated acids from acetic through octadecanoic were found separable on silicic acid-nitromethane chromatographic columns. The relative rates of travel with respect to that of the hexanoate derivative were determined and should be of assistance in the identification of small amounts of unknown organic acids.
T
HE identification of small amounts of organic acids present in the volatile aroma materials from grapes and wines has heen of interest in this laboratory for some time. The p-phenylphenacyl derivatives have proved valuable for this purpose when only a single acid or a mixture of acids of low molecular weight was present in a fraction. The chromatographic method for qeparation of mixtures of p-phenylphenacyl esters on silicic acid rolumns described by Kirchner, Prater, and Haagen-Smit (3) has permitted the separation and identification of these esters
when there is a considerable difference in molecular weight of the acid portion of the esters. Column chromatographic separations of p-phenylazophenacyl esters, as developed in this laboratory ( Z ) , have provided slightly better separations of the higher molecular weight acid derivatives. When only milligram quantities of p-phenylphenacyl ester are available, purification by either recrystallization or by chromatography is sometimes not effective enough to permit positive identification. The value of being able to prepare a second derivative from the small amount of pphenylphenacyl ester available in such cases is obvious. This paper reports the melting points and the chromatographic behavior of a number of the 2,4-dinitrophenylhydrazones of the pphenylphenacyl esters of the saturated fatty acids from acetic through stearic acids. MATERIALS
2,4-Dinitrophenylhydrazine Reagent. The reagent solution for preparation of the 2,4-dinitrophenylhydrazones of the p phenylphenacyl esters was made by dissolving 5.9 grams of 2,4dinitrophenylhydrazine in 130 ml. of reagent grade concentrated hvdrochlxic acid s3lution and then dilnting it with 870 ml. of 9570 ethyl alcohol. Aliquot8 of this stock solution \?ere filtered shortly before use. p-Phenylphenacyl Esters. Esters were prepared from- knon n
